The present invention relates to compositions comprising bacterial extracts useful for treating indications such as urinary tract or digestive disorders. The extracts may comprise bacterial lysates from cultures chosen from one or more species of Escherichia coli. In some embodiments, the extracts may comprise one or more species chosen from the following strains of E. coli: NCTC: 8603, 8621, 8622, 8623, 9026, 9111, 9119, 9707, and 9708 and I: 081, 082, 083, 084, 085, 086, 087, 088, and 089. Those strains are deposited under the Budapest Treaty. The strains indicated in the list with I-number were indexed by the Collection Nationale de Culture des Microorganismes at the Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, France. All of the other strains were indexed by the National Collection of Type Cultures in London.
In some embodiments, an extract is prepared from all of those strains. In other embodiments, only some of the strains are chosen. In some embodiments, for example, one or more strains is chosen from the “I” group and one or more strains is chosen from the “NCTC” group.
In some embodiments, one or more of the specific strains listed above may be omitted, or substituted with a different strain from E. coli, or from a different species of bacteria.
The extracts may be obtained by a process of alkaline lysis after cells are grown to a suitable optical density in a culture medium. In some embodiments, the bacteria are each grown on a medium that does not pose a risk of prion-related diseases or a risk of other diseases that may be transmitted through ingesting products obtained from animal-based media. For example, in some embodiments a vegetable-based medium is used to grow the cells, such as a soya-based medium. A synthetic medium may be used for cell growth in some embodiments, or a medium including biological extracts such as yeast extract and horse serum, which also do not pose such disease risks.
The lysates may also be filtered to remove nucleic acids and larger cellular debris. In consequence of the filtration, in some embodiments, the amount of nucleic acid present in the extracts is less than 100 μg/ml. In some embodiments, insolubilized compounds such as cell wall debris and insufficiently degraded lipopolysaccharide (LPS) are also removed by the filtration. Hence, in some embodiments, the resulting extract comprises soluble molecular components and does not contain significant amounts of insoluble or particulate material.
Saccharide components may be preserved in the extracts, including lipopolysaccharide (LPS) components. During the lysis process, saccharides may become chemically modified, for example, cleaved into smaller structures or substituted with other functional groups.
Racemization of amino acids during the lysis process also creates D-amino acids from the naturally occurring L-amino acids found in natural proteins. D-amino acids can be beneficial in increasing the time of effectiveness of the extracts, as they are not efficiently digested in the mammalian gut. Thus, antigenic molecules in the extracts that are chemically modified during lysis to contain D-amino acids remain in the patient's body for a longer time, allowing potentially for a stronger immunostimulating action.
While bacterial extracts have been used in the prior art to stimulate the immune system against digestive and urinary tract diseases, there has been a need to better standardize and control those extracts in order to make them safer, more effective, and longer lasting. For instance, it was previously thought that saccharide components, including potentially toxic lipopolysaccharide (LPS) components should be removed from bacterial extracts for safety reasons. (See, e.g., U.S. Pat. No. 5,424,287.) However, the instant invention provides a process that results in sufficient chemical modifications of LPS components that saccharides be safely retained. Retaining those components may improve efficacy as well by providing additional antigens.
For example, the inventors have discovered that monitoring the pH and the time of lysis allows for sufficient degradation of potentially allergenic or toxic cell wall components. Prior lysis conditions at lower pH's or shorter times, in contrast, produced extracts in which cell wall components and LPS were insufficiently chemically modified. (See, e.g., GB 2 054 374 A.) The resulting extracts were too allergenic to be safely administered to patients. In general, the inventors have discovered that products lysed at too low a pH and/or at too short a time had higher toxicity, lower protein extraction, and lower filterability.
In addition, the instant invention grows bacterial strains in culture media that do not pose disease risks, such as from prion diseases.
The filtration process can also influence the properties of the resulting extract in some cases, as the pore size of the filter, and sometimes, the chemical properties of the filter surface, alter the type of materials that are removed and retained. For example, the instant invention uses a filtration process that retains certain saccharides but removes other molecular components such as nucleic acids.
Thus, the instant invention provides parameters that standardize the bacterial extracts to help maintain consistent safety and efficacy.